Prolactin acts at multiple targets throughout the body, including the mammary gland, heart, liver, muscle and brain. Upon binding to its receptors, prolactin signals through the phosphorylation and thus activation of signal transducer and activator of transcription 5 (STAT5). There are two very similar STAT5 isoforms, termed STAT5a and STAT5b, which are selectively activated by prolactin in specific tissues. Various brain regions, including the hypothalamus, are prolactin responsive, although the STAT5 isoform involved in these actions is unknown. Immunohistochemical and western blot analysis were used to determine the expression and activation of STAT5a and STAT5b throughout the hypothalamus in adult wild-type and STAT5b-deficient mice. Both groups were pretreated with bromocriptine to suppress endogenous prolactin levels followed by the administration of ovine prolactin (10 mg/kg) for 45 min. STAT5a and STAT5b were expressed throughout the hypothalamus of wild-type mice. As expected, only STAT5a was detected in STAT5b-deficient mice, although, unexpectedly, there was a marked reduction in its expression compared to wild-type mice. When stimulated with prolactin, phosphorylated STAT5 was observed in the hypothalamus of wild-type but not STAT5b-deficient mice. By contrast, phosphorylated STAT5 was detected in mammary gland epithelial cells and adipocytes of STAT5b-deficient animals. Thus, although STAT5a was still expressed in the STAT5b-deficient mice, it was not phosphorylated in the hypothalamus in response to prolactin. These observations indicate that STAT5b but not STAT5a is the primary mediator of the action of prolactin in the hypothalamus. Despite the similarity between the two STAT5 isoforms, STAT5a was unable to compensate for the absence of STAT5b, suggesting that each isoform exhibits a unique biological activity.